Enterohemorrhagic E. coil (EHEC) O157:H7 is recognized as an important emerging pathogen responsible for producing hemorrhagic colitis and the hemolytic uremic syndrome (HUS) in humans. EHEC O157:H7 strains elaborate a potent Shiga toxin, which has been associated with the pathogenesis of HUS. No pili have yet been reproducibly identified in O157:H7 strains and therefore, it is still an enigma as to whether pili play a role in colonization of the intestine of their natural bovine or accidental human hosts. We have recently identified and purified a novel pilus structure produced by EHEC strain EDL933 and other O157:H7 strains. These pili are composed of an 18- kDa pilin subunit and its amino terminus shows identity to the predicted product of the ycbQ gene contained in the EDL933 chromosome. Sequence comparison analysis revealed that these pili, herein called YcbQ, belong to the virulence-associated pili family composed of F17, K99, and G pili found in several animal and human pathogenic E. coli strains, and CupA pili of Pseudomonas aeruginosa. Four genes (ycbQ, ycbR, ycbS, ycbT) with homology to F17 family piliation genes were identified by sequence homology in the genome of EDL933, being ycbQ the structural gene. Overall, the objective of this proposal is to advance knowledge of EHEC pathogenesis by elucidating the mechanism(s) of adherence of EHEC O157:H7 to human epithelial cells in culture. Several multidisciplinary approaches involving molecular biology, cell biology, ultrastructural analysis by high power electron microscopy, and biochemical and antigenic analysis will be carded out to extend our current knowledge on the interaction of EHEC O157:H7 with host target cells. The outcome of this proposal will provide important implications for diagnosis of EHEC disease and detection of O157:H7 in food sources and reservoirs. The information obtained will be important for prevention and control strategies of EHEC infections. The central focus of this proposal lies in the following specific aims: 1) To define the genes required for YcbQ pili biogenesis; 2) Define the role of YcbQ pili; and 3) Study transcriptional expression of ycb genes. [unreadable] [unreadable]